CN103814124A - In vitro cardiovascular model - Google Patents
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- CN103814124A CN103814124A CN201280030909.0A CN201280030909A CN103814124A CN 103814124 A CN103814124 A CN 103814124A CN 201280030909 A CN201280030909 A CN 201280030909A CN 103814124 A CN103814124 A CN 103814124A
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Abstract
The present invention relates to a tubule forming platform and an in vitro cardiovascular model for use in pharmacological studies. Furthermore, the invention relates to methods for the preparation said platform and model, and to a method of determining a biological activity of a test substance in said platform and cardiovascular model. Still further, the invention relates to an implantable cardiac structure for use in the treatment of cardiac disorders.
Description
Technical field
The present invention relates to form platform and external cardiovascular model for the tubule of pharmaceutical research.In addition, the present invention relates to the method for the preparation of described platform and model, and in described platform and cardiovascular model, measure the bioactive method of tested substance.In addition, the present invention relates to be used for the treatment of the implantable cardiac structure of heart disease.
Background technology
Cardiovascular systems belongs to important organ or system together with central nervous system with respiratory system, its function is very important for life.Therefore, must carry out cardiac toxic assessment to chemical substance for example medicine, industrial chemical, biocide, food and feed sanitas and makeup.These researchs mainly comprise the use of animal, are bad models but the test based on animal is proved to be the effect in the mankind for prediction conventionally.In addition, there is ethics problem in animal experiment, and cost is high and consuming time.For those reasons, EU Committee (European Commission) and both strategies of American regulation reason mechanism (US regulatory bodies) are in non-animal model, carry out safety testing, and test should be based in simulating human as far as possible closely the predictability human cell's organotypic model based on toxicity approach (" toxicity test of 21 century: prospect and strategy " (Toxicity Testing in the21st Century:A Vision and a Strategy), 2007) of condition.
Heart action potential abnormal, no matter by congenital sudden change or injured causing, can both cause the mankind's pathological symptom, particularly irregular pulse.The ADR of heart is very important, because they are normally serious and may be fatal, finds out as the various medicines of withdrawing from from market from the 1980's and generation nineteen ninety.These fatalitiess cause the attention of supervision department, and impel the ICH criterion S7B of announcement in 2005 and the generation of E14.These criterions make the proarrhythmia of studied all medicines tendentious non-clinical formalized with clinical assessment.Extend by drug-induced QT the second common cause that the arrhythmogenic causing is drug withdrawal, and caused increasing concern.QT interval, is subject to the impact of heart rate.The modal model using in the safety pharmacology research of novel drugs is at present the heart that separates from cavy or rabbit of animal model and containing or the isolated model from the Purkinje cell of dog separation.There is not the external heart model of the empirical tests that can be used for these objects.
Several different external 3D heart tissue construction (Zimmermann etc., Circulation Research, 2002,90:22 with retractable property and action potential are developed; Akiyama etc., Int.J.Mol.Sci., 2010,11:2910).The shortcoming of existing research model is that they are based on zoobiology (rat cell), and model can only maintain function in the short period of time (a couple of days).Therefore can only assess short term effect.Therefore, in order to simulate the heart function relevant from the mankind (cardiac frequency, heartbeat intensity, electroactive, different channel activity), the functional organization's construction based on human cell that needs exploitation to there is associated biomolecule mark and physical and chemical condition control and maintain.
US2009/0169521 discloses a kind of artificial 3D cardiac structure that is used for the treatment of heart disease.Described structure is by being seeded in myocardial cell, endotheliocyte and inoblast in artificial scaffolds or altogether and being seeded in artificial scaffolds and obtaining altogether.Due to the cell assembling (Norotte etc. in exogenous support possibility interference cell-cell interaction and multilayer tissue's construction, Biomaterials, 2009,30:5910), therefore disclosed cardiac structure is not best for the application in pharma-toxicology research.
Except the toxicity research of chemical substance and biological substance, organotypic cardiac module that also need to be based on human cell in the research of the novel drugs for cardiovascular disorder.In western countries, cardiovascular disorder is the modal cause of death, and wherein heart failure is one of common disease.Therefore there is active demand in the medicine to exploitation reparation heart function and organizational project treatment.One method is to repair infarcted region with stem cell therapy.The object of stem cell therapy is to make patient's self differentiation of stem cells become to have the myocardial cell of function, and transplants described cell to repair myocardium affected area.But, before stem cell therapy can be applied to clinical use, need more research to understand better differentiation, propagation and the behavior of stem cell.Organizational project treatment will greatly be benefited from the functional cardiac module based on human cell that comprises that blood vessel is supported.
Therefore, exist confirmed demand at the heart external model for empirical tests in the art.
Summary of the invention
On the one hand, the invention provides a kind of external cardiovascular structures, the tubule that it comprises separation forms platform and myocardial cell.Described tubule forms platform and comprises mankind's fat stem cell (hASC), and does not optionally contain the biomaterial of any exogenous substrates component or interpolation.In some embodiments, described platform also comprises the endotheliocyte that forms tubule, for example the endotheliocyte in the multipotential stem cell source of the endotheliocyte in the endotheliocyte in human umbilical vein endothelial cell, human microvascular endothelial cell, mankind's fat stem cell source, hESC source, induction, endotheliocyte, the endothelial progenitor cells in transdifferentiation source or the endotheliocyte obtaining by genetic modification.
On the other hand, the invention provides one and be used for the treatment of cardiopathic external cardiovascular structures.
On the other hand, the invention provides the tubule formation platform of separation as above.
On the other hand, the invention provides a kind of method of producing tubule formation platform.Described method comprises the following steps: a) to provide hASC; B) augment in the serum-free perfect medium of VEGF and FGF-2, optionally under the condition of biomaterial that does not have any exogenous substrates component or interpolation, cultivate described hASC.In some embodiments, described method also comprises provides the step that forms the endotheliocyte of tubule and they and described hASC are carried out to common cultivation.
On the other hand, the invention provides a kind of method of producing external cardiovascular structures as above.Described method comprises the following steps: a) to provide the endotheliocyte of hASC, myocardial cell and optional formation tubule; B) cultivate described hASC, optionally together with the endotheliocyte of described hASC and described formation tubule, cultivate; On the top of the culture c) described myocardial cell being formed in step b), cultivate; And d) use VEGF and FGF-2 to the cell culture forming in step c).
In addition, one aspect of the present invention relates to a kind of bioactive method of measuring tested substance.Described method comprises the following steps: a) to provide tubule as above to form platform or external cardiovascular structures; B) use described tested substance to described platform or structure; C) in described platform or structure, measure the effect of described tested substance; And d) by the effect of measuring in step c) with do not exist the respective effects of measuring under the condition of described tested substance to compare.In some embodiments, biological activity to be determined is selected from cytotoxicity, tubule and forms and regulate the speed, for example mycardial contractility power of mechanical properties of active, for example mycardial contractility of electrical properties or/and basic cellular metabolism.
Another aspect of the present invention provides a kind of cardiopathic method for the treatment of in the patient of needs, and described method comprises above-mentioned cardiac structure is implanted in described patient.Described cardiopathic limiting examples can be selected from coronary heart disease and DCM (dilated cardiomyopathy).
Other features of the present invention, specific implementations, object, details and advantage are illustrated in dependent claims, figure, detailed description and embodiment below.
Accompanying drawing explanation
Below with reference to the accompanying drawings, utilize preferred implementation to be described in more detail the present invention, in described accompanying drawing
Fig. 1 shows the photo of the tubule formation of the mono-culture of hASC and hASC+HUVEC coculture.Anti-von Willebrand for cell (Sigma, red fluorescence is illustrated by the second antibody of TRITC coupling, 1:100, Sigma for anti-angiogenic property christmas factor antibody, 1:500) is dyeed.The comparison that the tubule of the mono-culture of Figure 1A: hASC and hASC+HUVEC coculture forms.Cell is cultivated in the EGM-2BulletKit substratum that is rich in somatomedin to also induction of vascular and generated 3 or 6 days.Figure 1B: the semi-quantitative analysis that the tubule between different treatment forms.At 3 and 6 days, mono-hASC culture and hASC+HUVEC coculture are compared mutually.Sxemiquantitative scale is according to (Front.Pharmacol, 2011,1:147.) such as Sarkanen.Result is reported as mean value ± SD, and in the time of p<0.05*, p<0.01** and p<0.001***, it is significant that difference is considered to.Fig. 1 C: for contrast, by HUVEC with 4000 cell/cm
2bed board is also grown in the EGM-2 that is rich in somatomedin, and the hASC+HUVEC coculture of growing under the condition of not exogenous interpolation somatomedin.Fig. 1 D: hASC+HUVEC is cultivated in containing human serum (EGM-2,2%HS) or not containing the EGM-2 that is rich in somatomedin of serum (EGM-2 serum-free).
Fig. 2 shows and is carrying out after vasculogenesis induction with the EGM-2 substratum that is rich in somatomedin, the photo of the expression of pericyte and smooth muscle cell differentiation mark in Minute Tubule Structures.Form in order to detect tubule, anti-von Willebrand for cell culture (Sigma, red fluorescence is illustrated by the second antibody of TRITC coupling, 1:100, Sigma for anti-angiogenic property christmas factor antibody, 1:500) is carried out to immunostaining.In order to detect tubule maturation, by anti-α SMA antibody (1:200 for culture, Sigma), anti-COLIV antibody (1:500, Sigma), anti-PDGFR β antibody (1:500, Sigma), anti-SMMHC antibody (1:800, Sigma) or anti-calponin antibody (1:800, Sigma) carry out immunostaining, all these green fluorescences are all illustrated by the second antibody (1:100, Sigma) of FITC coupling.The image illustrating is all the merging image of the dual immunofluorescence of the 6th day, what make an exception is the image of anti-PDGFR β antibody, it is the merging image of the 3rd day, and the image of anti-COLIV antibody and anti-calponin antibody, two of second antibody-anti-COLIV antibody/anti-calponin antibody staining (large image) that show dyeing (little image) and FITC coupling for them merge images.
Fig. 3 shows the photo of the external cardiovascular model based on hASC, HUVEC and neonatal cardiac myocytes of cultivating 10 days.Scale bar is 100 μ m.Form in order to detect tubule, anti-von Willebrand for cell culture (anti-angiogenic property christmas factor antibody, 1:500, Sigma, the second antibody of TRITC coupling, 1:100, Sigma) is carried out to immunostaining.In order to detect myocardial cell, culture is carried out to immunostaining by the second antibody (1:100, Sigma) of heart specificity anti-troponin T antibody (1:500, Abcam) and FITC coupling.
Fig. 4 shows the photo of the myocardial cell's based on hASC, HUVEC and hESC source who cultivates 10 days external cardiovascular model.Scale bar is 100 μ m.Form in order to detect tubule, anti-von Willebrand for cell culture (anti-angiogenic property christmas factor antibody, 1:500, Sigma, the second antibody of TRITC coupling, 1:100, Sigma) is carried out to immunostaining.In order to detect myocardial cell, culture is carried out to immunostaining by the second antibody (1:100, Sigma) of heart specificity anti-troponin T antibody (1:500, Abcam) and FITC coupling.
Fig. 5 is multiple electrode array (MEA) record, and it shows the electrical signal that comes from the cardiovascular model of synchronous after model construction for 4 days.
Concrete facts mode
The invention provides the external cardiovascular structures using in pharmacology security and toxicity research, i.e. cardiovascular model.Described model comprises the myocardial cell who is cultured on tubule formation platform.It is shocking, can under the condition of biomaterial that does not use any exogenous substrates or interpolation, obtain such functional angiocarpy model.
Be generally used for producing exogenous support possibility interference cell-cell interaction and the cell assembling of multilayer tissue's construction.Therefore, thus, of the present invention is favourable without the cardiovascular model of support.Best tissue constructs should not contain component or non-natural timbering material of animal-origin, and should be only containing natural somatomedin and the protein being present in tissue.At least in some embodiments, cardiovascular model of the present invention meets these requirements, and there is the feature of ripe blood vessel,, except forming Minute Tubule Structures, model also has following characteristics: pericyte is raised, basement membrane forms and the formation of the vessel support layer of smooth muscle cell.
In the time using in this article, term " comprise " contain term " by ... form " and " substantially by ... formation ".
In the time using in this article, term " tubule formation platform " refers to the multi-layer cellular structure with the ability that is self-assembled into blood vessel network structure.
In one embodiment, tubule formation platform can only for example, be formed by adipose-derived matrix/stem cell (ASC) mankind's fat stem cell (hASC) structure.Such tubule forms platform and is called as single culture model.
In the time using in this article, term " adipose-derived stem cell " or " ASC " refer to the unsorted matrix blood vessel fraction obtaining from fatty tissue.Such fraction is heterogeneous, and comprises mescenchymal stem cell.According to nearest term, " ASC " also can be called as " adipose-derived stroma cell ".Obtain mankind ASC(hASC) method be easily to obtain in the art, include but not limited to disclosed method in embodiment 1.As known in the art, ASC has the ability that is divided into various kinds of cell type.
In another embodiment, by carrying out common cultivation and construct tubule and form platform forming the endotheliocyte of tubule and hASC.Such tubule forms platform and is called as coculture model.
In the time using in this article, term " forms the endotheliocyte of tubule " and refers to the endotheliocyte with the ability that forms for example blood vessel network of blood vessel structure.The limiting examples of endotheliocyte that forms tubule comprises the endotheliocyte in the multipotential stem cell source of endotheliocyte, the induction in the endotheliocyte in human umbilical vein endothelial cell (HUVEC), human microvascular endothelial cell, mankind's fat stem cell source, hESC source, the endotheliocyte in transdifferentiation source, the endotheliocyte that comes from the endothelial progenitor cells of its hetero-organization and obtain by genetic modification.For the measure of inducing the endothelium of above-mentioned cell to break up, easily know to those skilled in the art.The endotheliocyte that forms tubule is also commercially available.
Embryonic stem cell (ESC) is to have to be divided into the pluripotent cell of the ability of for example endotheliocyte of various different cell types widely.The method that obtains embryonic stem cell is easily to obtain in the art.In addition, WO2007/130664 discloses a kind of for obtain hESC's novel method likely in the situation that not damaging donor embryo, and the method is called as blastomere biopsy.
In the time using in this article, term " multipotential stem cell of induction " (iPSC) refers to by growing reprogrammed (developmental reprogramming) from noble cells, normally from becoming the human body cell multipotential stem cell that for example inoblast produces.Such cell has for example been described in WO2008/151058 and US2008/076176.The human pluripotent stem cell of induction is called as hiPSC.
In the time using in this article, term " transdifferentiation " or " pedigree reprogrammed (lineage reprogramming) " refer to the multipotency state of a kind of mature cell type in the middle of not experiencing or progenitor cell type and are transformed into another kind of mature cell type.
Tubule forms platform and can obtain by following method, in described method, by i) ASC or ii) ASC and the endotheliocyte that forms tubule, be layered in the cell culture medium of the support endothelial cell growth on Tissue Culture Plate or tissue culturing plate for example 24 holes, 48 holes, 96 orifice plates or microwell plate.The limiting examples of such substratum is the EGM-2BulletKit that can obtain from Lonza
tM.Can be in any endothelial growth substratum (being supplied by for example Lonza, Provitro, Promocell, BD), or containing the lower concentration mankind or animal serum or containing serum and contain bFGF, VEGF, xitix, heparin and/or hydrocortisone as DMEM, the DMEM/F12 of enriching substance or knock out (KO) DMEM(for example supplied by Gibco Invitrogen, Sigma, BD, Lonza) in induce tubule to form.The limiting examples of the optional factor that can further comprise is such as triiodothyronine of Regular Insulin, IGF-I, hEGF, Transferrins,iron complexes and/or hormone.
In some embodiments, the basal surface of Tissue Culture Plate can have groove or cut, so that the tubule that will form aligns towards required direction.
In single culture model, conventionally but not necessarily by cell with about 24x10
4individual cell/cm
2or higher density is carried out bed board.In coculture model, conventionally but not necessarily by ASC and endotheliocyte with 2:1 to 8:1, preferably the ratio of 5:1 is carried out respectively bed board.In one embodiment, by ASC with about 20x10
4individual cell/cm
2density carry out bed board, and will form the endotheliocyte of tubule with about 4x10
4individual cell/cm
2density carry out bed board.In some embodiments, for example HUVEC of cell that forms tubule is carried out to bed board for late 1 to 3 hour than for example hASC of ASC.
Cardiovascular model of the present invention obtains by following method, in described method, myocardial cell is inoculated or is layered on the top of tubule formation platform, moulds thus the mankind or animal hearts.The limiting examples that is applicable to the myocardial cell of this model comprises the myocardial cell in human pluripotent stem cell (hiPSC) source of the myocardial cell in neonatal cardiac myocytes, hESC (hESC) source, induction, the myocardial cell in adult stem cell source, myocardial cell and human myocardium's cell in mankind's transdifferentiation source.For inducing the measure of Cardiomyocyte Differentiation to be well known in the art, and include but not limited to by (Circulation such as Mummery, 2003,107:2733) the differentiation of the endoderm cell of exploitation induction, by (NatBiotechnol, 2007,25(9) such as Laflamme: the 1015) differentiation of the activin A of exploitation and BMP4 induction, and the embryoid technology of being developed by (Circ.Res.2002,91:659) such as Kehat.
In some embodiments, myocardial cell is laid in serum-free perfect medium (CSFM) on the top of tubule formation platform.This is specially adapted to rat myocardial cell.The limiting examples that is suitable as the commercially available cell culture medium of the basic medium of CSFM is the DMEM that can obtain from Gibco
tMor DMEM/F12 or knock out (KO) DMEM.
If realize cardiovascular structures with mankind myocardial cell, substratum can be serum-free or contain bovine serum or the human serum up to approximately 10%.In such circumstances, for example DMEM/F12 can be used as basic medium.
Myocardial cell is conventionally with about 1x10
5or about 2x10
5individual cell/cm
2density carry out bed board, but inoculum density is not limited to these values.
Conventionally but not necessarily, myocardial cell is seeded on the top of tubule formation platform than the cell that forms tubule and form platform for late one day.For cardiovascular model, before inoculation myocardial cell, being completed into tubule is not prerequisite.
For aliging of the formation of real-time tracing tubule and myocardial cell and tubule, can utilize for example slow virus infection, by for example green or yellow fluorescence protein are inserted in endothelial cell gene group, the endotheliocyte and/or the myocardial cell that form tubule are carried out to fluorescent mark.Also can carry out genetic modification (comprise and insert reporter gene, disease specific gene, differentiation associated gene) to cell.
Conventionally, by myocardial cell's bed board one day after, use vascular endothelial growth factor (VEGF) and Prostatropin (FGF-2) so that induction tubule forms to cell.This fresh CSFM that can augment by substratum is replaced to described somatomedin carries out.Conventionally, use at about 1ng/ml to about 20ng/ml, the VEGF in 10 to 15ng/ml concentration range preferably; And conventionally use at about 0.5ng/ml to 2ng/ml, the FGF-2 in 1 to 2ng/ml concentration range preferably.
Include but not limited to xitix, heparin, hydrocortisone, type-1 insulin like growth factor (IGF-1), Urogastron (EGF), be preferably mankind EGF for the optional reagent that improves vasculogenesis induction at model, and any combination.
Be suitable for other optional medium components that in model of the present invention induction of vascular generates (being that tubule forms) and comprise that concentration is up to 10% bovine serum and human serum, and bovine albumin and human albumin.Hold intelligible as professional and technical personnel, for the production of and/or any substratum of utilizing tubule of the present invention to form platform and/or cardiovascular model can contain normally used any composition, for example microbiotic, L-glutaminate and Sodium.alpha.-ketopropionate in cell culture medium.
In cardiovascular model, myocardial cell is to keep survival and to have function than the possible in the past longer time.In some embodiments, the shrinkability of neonatal cardiac myocytes can maintain three weeks, and in contrast to this, the myocardial cell in hiPSC and hESC source can maintain the several months.In cardiovascular model, myocardial cell's viability and shrinkability are more important than the Minute Tubule Structures being completed into.Therefore,, according to various embodiments of the present invention, can test tested substance with the cardiovascular model with appropriate tubule formation.
In some embodiments, using VEGF and FGF-2 one day after, adding the material that will test to cell in cardiovascular model of the present invention.Prerequisite is that myocardial cell must have functional property before adding chemical substance.If needed, the effect of described material can be tracked for example 2 to 3 weeks or several months even, this depends on myocardial cell's application and source.
The example of biological effect to be determined includes but not limited to toxic effect, and increase or reduction that it is for example expressed by assessment different genes are measured; The viability of cell, it for example, is measured by means of different (MTT test, Neutral red uptake (NRU) assay method or the LiveDead that can obtain from Invitrogen measure); For example mycardial contractility speed of electrical properties and the variation of repolarization time or arrhythmia event, it is for example measured by measuring QT interval; The variation of for example convergent force of mechanical properties, it is measured by different plane biosensor or traction; Cardiac marker is for example for detection of the connection protein-4 3 of GAP node or such as the immunostaining of the mark of specific heart TnT; And the variation of cellular metabolism (variation, glucose consumption, oxygen consumption and the release of carbonate dioxide of for example lactic acid formation, calcium flux, ionic channel).These effects can be with any required combination dividually, sequentially, assess concurrently or side by side.
Cardiovascular model can contain for example plane biosensor of one or more sensors, for assessment of any cytological effect above-mentioned.Applicable sensor includes but not limited to electrochemistry, electricity and/or optical pickocff.If can comprise for monitor and needs, for regulating other sensors of physicochemical property of substratum.
In some embodiments, tubule forms the vasculogenesis character that platform itself can be used for assessing tested substance.The limiting examples of vasculogenesis character to be assessed comprises that tubule forms ability (for example, by measuring little length of tube and/or branch, or the close-connected existence of definite endothelium) and the ripe ability of tubule (for example by determining that basement membrane forms, the existence that serves as a contrast of the pericyte of ripe Minute Tubule Structures and smooth muscle cell).In such circumstances, do not add myocardial cell to cell culture.Can for example use or not use above-mentioned optional angiogenesis inducing agents to carry out vasculogenesis induction one day after by VEGF and FGF-2, form platform to tubule and apply tested substance.Vasculogenesis character can tracked for example a couple of days or two weeks.Even can, before tubule is completed into, apply tested substance to model.
The limiting examples of the tested substance that will screen in cardiovascular and angiogenesis model of the present invention comprises chemistry and biological substance for example micromolecular compound, nanoparticle, polypeptide, antibody and somatomedin.
Although under the condition of biomaterial that does not have any exogenous substrates component and interpolation, it is functional for the object of pharmacology security and toxicity test that cardiovascular structures and tubule form platform, and simulate heart tissue and there is no interfering non-origin component, but in some cases, such component being included in may be favourable in model and/or platform.Such embodiment can be used for for example in artificial heart construction, testing security and the toxicity of tested substance.Will be provided at that cardiovascular model and/or tubule form applicable exogenous substrates component in platform or the limiting examples of biomaterial includes but not limited to synthetic or for example collagen protein I of natural polymer or IV, hyaluronic acid, gelatin or other extracellular matrix components.
In some situation of the present invention, can build the cardiovascular structures of the biomaterial that contains exogenous substrates component and/or interpolation as implantable 3D cardiac structure, be used for the treatment of heart trouble, include but not limited to coronary heart disease and DCM (dilated cardiomyopathy).
In the time using in this article, term " treatment " not only refers to the healing completely of disease, but also refers to prevention, alleviation and the improvement of disease or relative symptom.
For therapeutic purpose, importantly cardiac structure is not contain foreign matter, and it does not contain any component obtaining from external source, or is not to prepare under the condition that contains external reagent.In addition,, for therapeutic purpose, it may be favourable using autogenous cell.
It will be apparent to one skilled in the art that along with technical progress, concept of the present invention can be implemented with various different modes.The present invention and embodiment thereof are not limited to above-described example, but can in the scope of claims, change.
Embodiment
All working carries out according to the guilding principle of the national ethics council.Mankind's umbilical cord obtains from the cesarean section of plan, and human adipose tissue sample obtains from surgical operation, has both obtained suitable license and informed consent from Tampere university hospital.In addition, Pirkanmaa hospital area Ethics Committee approved the obtaining and using of mankind iPS and hESC cell, license number is respectively R08070 and R051116.
Embodiment 1. forms structure and the sign of platform based on the tubule of coculture
Human umbilical vein endothelial cell's (HUVEC) separation:
HUVEC cell stems from the umbilical cord obtaining from the cesarean section of plan, described cesarean section has the informed consent (license number R08028 comes from the Pirkanmaa hospital area Ethics Committee of the Tampere of Finland) that comes from Tampere university hospital.Carry out the separation of huve cell (HUVEC) from mankind's umbilical vein according to the description of (J Clin Invest, 1973,52:2745) such as Jaffe, but method has been carried out to further optimization.Separate umbilical cord from placenta, and umbilical vein is carried out to intubate with 20G syringe needle.Make syringe needle firm by clamp umbilical cord with surgical clip above syringe needle.With umbilical cord buffered soln (UBS; 0.1M phosphate buffer soln, contains 0.14M NaCl, 0.004M KCl and 0.011M glucose) pour into vein to wash blood off, then clamp the other end of umbilical vein with surgical clip.Vein is inculcated with 0.05% collagenase I.By umbilical cord in water-bath at 37 ℃ incubation reach 20min.After incubation, by with PBS perfusion, the collagenase I solution that contains HUVEC is flushed to 50ml polypropylene tube (Sarstedt) from umbilical cord.Cell, with the centrifugal 10min of 200xg, is washed once with substratum, and recentrifuge, is resuspended in EGM-2BulletKit substratum (Lonza Group Ltd, Basel, Switzerland), and is inoculated into 75cm
2in bottle.By cell at 5%CO
2in incubator, at 37 ℃, cultivate.Within every 2 to 3 days, change substratum, and in the time converging, cell is separated.For measure contrast, by HUVEC with 4000 cell/cm
2carry out bed board, and cultivate in EGM-2BulletKit substratum.
Examine under a microscope morphology, cell culture purity and the cell proliferation of the HUVEC of separation every day.Within every 2-3 days, change substratum.In the time converging, use Tryple Express by cellular segregation.To there is the pure HUVEC culture of good multiplication capacity, when former culture (p0) with the cultivation of going down to posterity of the ratio of 1:2 – 1:4, in 1st generation (p1) and later with the cultivation of going down to posterity of the ratio of 1:3 – 1:5.
Slow virus infection:
Slow virus construction pLKO-MISSION-Bright-GFP is purchased from Biomedicum Genomics(BMGen, Biomedicum Helsinki, Helsinki, Finland).Use 300 μ l pLKO-MISSION-Bright-GFP(1U/ml in 1mlEGM-2Bullet Kit substratum) HUVEC of low passage number is infected.Use 8 μ g/ml hexadimethrine bromides (Sigma) to accelerate virus infection.After 24 hours, substratum is replaced with to fresh EGM-2 substratum at incubation.Use clone's ring to select high fluorescence clone, and use dilution cloning to carry out further selecting to obtain pure GFP-HUVEC culture.After the HUVEC infecting is increased, they are used to hASC as described below and HUVEC coculture assay method.
The separation of mankind's fat stem cell (hASC):
According to previously described (Gimble and Guilak, Cytotherapy, 2003,5:362; Hong etc., Mol Cell Biochem, 2005,276; Niemela etc., J Craniofac Surg, 2007,18:325-335) carry out stem cell separable programming.In simple terms, human adipose tissue sample is cut into small pieces, with the Eagle substratum nutritional blend F-12(DMEM/F12 of Dulbecco improvement, Gibco, Invitrogen, Carlsbad, CA, USA) in 0.05% collagenase I(Invitrogen, Paisley, Scotland, UK), in convolution water-bath, at 37 ℃, carry out enzymic digestion 60min.By being organized under room temperature (RT) with the centrifugal 10min of 600x g of digestion.The tissue filter of digestion is by 100 μ m filters (Sarstedt, N ü mbrecht, Germany), centrifugal, and filter by 40 μ m filters (Sarstedt).At 75cm
2bottle (Nunc EasyFlask
tM, Nunc, Roskilde, Denmark) in, cell is seeded in and has augmented 1%L-glutamine (L-glut, Gibco), 1% microbiotic-anti-mycotic agent mixture (AB/AM, Gibco) and 15% human serum (HS, Cambrex, East Rutherford, NJ, USA) DMEM/F12 in.Second day, by PBS washing several for cell.Cell is maintained to 37 ℃, 5%CO
2under air atmosphere and constant humidity, and within every 2 to 3 days, change substratum.After growing to and converging, by cell with the ratio of 1:2-1:3 separately, or be further used for cell culture studies.
The common cultivation of hASC and HUVEC:
Mankind ASC(is gone down to posterity at most 7 times) with 20000 cell/cm
2density be seeded in 48 orifice plate (Nunclon
tMmultidishes, Nunc, Roskilde, Denmark) in EGM-2BulletKit(Lonza) in substratum.As above the HUVEC(cultivating is gone down to posterity at most 4 times) immediately with 4000 cell/cm
2density be seeded in carefully on the top of hASC.After bed board second day, applies VEGF(10ng/ml to coculture) and FGF-2(1ng/ml).
By cell cultures 3 or 6 days, then carry out immunocytochemistry or quantitative RT-PCR.Change substratum, and the cell of cultivating 3 days is applied to primary treatment, apply twice processing to cultivating the cell of 6 days.
Immunocytochemistry:
Use the endothelial cell specific antibodies (the anti-vWf first antibody producing in rabbit, 1:500, Sigma) for vWF ELISA, to tubule, formation is carried out visual.For evaluator class fat differentiation of stem cells, carry out parallel dual immunofluorescence dyeing with α-vWf.Will be for common pericyte mark α-smooth muscle actin (monoclonal anti-α SMA clone 1A4, 1:200, Sigma), vascular smooth muscle cell mark unstriated muscle myoglobulin heavy chain (anti-SMMHC antibody, 1:800, Sigma), the anti-calponin antibody of shrinkability smooth muscle cell mark calponin(, 1:800, Sigma), pericyte and smooth muscle cell progenitor cell marker thing platelet derived growth factor receptor-β (anti-PDGFR β antibody, 1:800) or the anti-COLIV antibody of basement membrane mark collagen protein IV(, 1:500, Sigma) first antibody and the combination of anti-vWf antibody.By PBS washing for cell 3 times, fix 20 minutes, use 0.5%Triton X-100(JT Baker with 70% ice-cold ethanol, Phillipsburg, NJ, USA) permeabilized 15 minutes, and with 10% bovine serum albumin (BSA, Sigma) sealing unspecific staining 30 minutes.After sealing, by cell and first antibody to incubation under RT 1 hour.By PBS washing three times for cell, with second antibody incubation 30min, described second antibody is Anti-TNF-α rabbit igg TRITC antibody (1:100 for anti-vWf antibody, Sigma), Anti-TNF-α mouse IgG FITC antibody (1:100, Sigma) for anti-α SMA antibody, anti-COLIV antibody, anti-PDGFR-β antibody and anti-SMMHC antibody.By nucleus Hoechst33258(1ug/ml, Sigma) dyeing 5 minutes with PBS washing 5 times.For anti-GFP dyeing, first antibody is to being the mouse monoclonal antibody (Abcam for GFP, Cambridge, UK, 1:100) and anti-vWf antibody, second antibody is respectively anti-mouse IgG TRITC antibody (Sigma, 1:100) with for polyclonal antibody (the Acris Antibodies GmbH of rabbit igg FITC, Hiddenhausen, Germany, 1:500).Use Nikon Eclipse Ti-S microscope (Nikon, Tokyo, Japan) by fluorescent visual, and with Adobe Photoshop software 7.0(Adobe Systems, San Jose, CA, and Corel Draw software 10.0(Corel Corporation USA), Ottawa, ON, Canada) processing image.
The microscopical analysis that tubule forms:
After immunocytochemical stain, use Nikon Eclipse TS100 microscope (Nikon, Tokyo, Japan) to analyze tubule with 40x magnification from the hole of 48 orifice plates.Use from 0 to 10 sxemiquantitative grading scale to carry out quantitatively the degree of tubule different cultures, described classification is based on tubule formation, little length of tube and branch, as (Sarkanen etc., 2011) described in the research before us.
Statistical analysis:
Carry out statistical analysis, and use GraphPadPrism5.0(GraphPad software, Inc., San Diego, CA, USA) processing chart.Tubule is formed and RT-PCR result is carried out single factor ANOVA analysis, and carry out Dunnett ' s subsequently and Bonferroni ' s checks at where applicable afterwards.Result is reported as mean value ± SD, and in the time of p<0.05*, p<0.01** and p<0.001***, it is significant that difference is considered to.
Result:
In two different time points (the 3rd day and the 6th day), tubule formation ability and the anti-vWf antibody positive endothelium Minute Tubule Structures of coculture are assessed and compared.Coculture demonstrates in early days (the 3rd day) tubule network and forms, and described tubule network formation is repeatably and does not rely on clone or the passage number of cell.At the 6th day, coculture demonstrated the multiplication rate of very accelerating, because there is the multilayer blood vessel network of a large amount of densifications to form.
The sxemiquantitative assessment that tubule forms shows that coculture had the significantly more tubule (p<0.001) than the 3rd day at the 6th day.The compared with control cells of growing under the condition that there is no somatomedin and be rich in the independent HUVEC growing in the EGM-2 substratum of somatomedin, only demonstrates respectively a small amount of tubule and forms or do not have tubule to form.
Also coculture is carried out to immunocytochemical stain.At the 3rd day, the expression of PDGFR β was the strongest, and as being observed around the dots structure of developmental tubule all the time.At the 6th day, to a certain degree to observe PDGFR β.The COLIV that demonstrates basement membrane growth is expressed very widely in coculture.Described expression and developmental tubule are located altogether, cover described tubule.At the 6th day, α-SMA and SMMHC positive cell wide expression in coculture, be usually located in the tapping point of Minute Tubule Structures and between tubule.Between the 3rd day and the 6th day, SMMHC expresses increase.Can reach a conclusion, coculture model forms fine and close multilayer blood vessel network, the smooth muscle cell with retractable property that for example complete basement membrane of its character with ripe blood vessel forms and aligns with tubule.More ripe compared with any angiogenesis model of this coculture model and former exploitation.
Obtain as described in example 1 above mankind ASC, and by it with 20000 cell/cm
2density be seeded in 48 orifice plate (Nunclon
tMmultidishes, Nunc, Roskilde, Denmark) in EGM-2BulletKit substratum in.Cell, in the commercially available substratum that is rich in somatomedin of this EGF of containing of EGM-2BulletKit substratum, VEGF, bFGF, IGF-I, xitix, heparin, 0.1% gentamicin/amphotericin B and 2%FBS, or is cultivated 3 or 6 days in the DMEM/F-12 substratum of augmenting 15%HS, 1mM L-glut and 1%AB/AM.Change substratum, and apply primary treatment to the cell of cultivating 3 days, apply twice processing to the cell of cultivating 6 days.As measuring contrast, hASC is cultivated in the DMEM/F-12 substratum of augmenting 15%HS, 1mM L-glut and 1%AB/AM.
Result:
In the mono-culture of hASC, be not so good as in coculture strong like that to the induction of vasculogenesis.But, in single culture, usually observe pericyte and the smooth muscle cell mark of support blood vessels.
Structure and the sign of embodiment 3. external cardiovascular models
Obtain as described in example 1 above the HUVEC and the hASC that use in the present embodiment.Neonatal cardiac myocytes is taken from the neonate rat young baby of 2 to 3 ages in days.
As described below in 48 orifice plates construct unfaithful intention vascular pattern:
The 0th day: tubule forms the structure of platform
Coculture model: hASC(is gone down to posterity at most 4 times) with 20000 cell/cm
2density be seeded in the EGM-2BulletKit substratum in 48 orifice plates.After 1-3 hour, maximum the HUVEC(in EGM-2 substratum 4 times are gone down to posterity) with 4000 cell/cm
2density be seeded in carefully on the top of hASC.
Single culture model: hASC(is gone down to posterity at most 4 times) with 24000 cell/cm
2density be seeded in the EGM-2BulletKit substratum in 48 orifice plates.
The 1st day: the structure of external cardiovascular model
Neonatal cardiac myocytes in serum-free perfect medium (CSFM) (100000,200000 or 40000 cells) is seeded on the top of tubule formation platform.
The 2nd day: the induction of differentiation
Substratum is replaced by the CSFM that augments 10ng/ml vascular endothelial growth factor (VEGF) and 1ng/ml Prostatropin (FGF-2).Time substratum is replaced with to fresh culture on every Wendesdays.
Result:
Live and contractile neonatal cardiac myocytes is survived approximately 7 days in single culture model, survival at least 14 days (referring to table 1) in coculture model.In whole incubation time, maintain the cellular form of striated form.Myocardial cell is along Minute Tubule Structures or near Minute Tubule Structures orientation, and in whole culture synchronous.
The shrinkability of table 1. neonatal cardiac myocytes
* the approximation obtaining by visual inspection
(EGM-2BulletKit Single Quots enlargement, Lonza) and heparin (EGM-2Single Quots enlargement, Lonza).
Substratum 3: augmented 2%FBS(foetal calf serum, Gibco), the CSFM of 10ng/ml VEGF and 1ng/ml FGF-2.
Substratum 4: vasculogenesis stimulates substratum: the endothelial basal medium (EBM-2, Lonza) of having augmented 10ng/ml VEGF, 1ng/mlFGF-2,0.1% gentamicin (GA-1000, Lonza), 2% foetal calf serum and 1mM L-glutaminate.
Substratum 5: vasculogenesis stimulates substratum+human serum: augmented 10ng/ml vascular endothelial growth factor and 1ng/ml Prostatropin (FGF-2, Sigma), 0.1% gentamicin (GA-1000, the endothelial basal medium (EBM-2, Lonza) of Lonza) and 2% human serum (Lonza) and 1mM L-glutaminate.
Embodiment 4. comparative results
Form in platform at the tubule based on coculture, the tubule of assessing neonatal cardiac myocytes (NRC) under 7 kinds of different treatment forms and mycardial contractility ability.
Substratum 1: augmented 10ng/ml VEGF(Sigma Aldrich, Manassas, VA, USA) and 1ng/ml FGF-2(Sigma) CSFM(serum-free perfect medium).
50ml CSFM is made up of following ingredients:
-DMEM/F-12 42ml
-200mM L-glutaminate 0.64ml
-100x penicillin/streptomycin 0.5ml
-0.1nM T3 0.5μl
-10x BSA 5ml
-100mM Sodium.alpha.-ketopropionate 1.4ml
-ITS 0.576ml
Substratum 2: the CSFM that has augmented 10ng/ml VEGF, 1ng/ml FGF-2, xitix (EGM-2Single Quots enlargement, Lonza), hydrocortisone.
Substratum 6:EGM-2BulletKit substratum (Lonza), wherein replaces 2% foetal calf serum (Lonza) with 2% human serum (Lonza).
Substratum 7: the EGM-2BulletKit substratum (Lonza) that does not contain foetal calf serum.
Table 2. mycardial contractility ability and tubule form
* by visual inspection
* is according to Sarkanen etc., the scale of 2011 1-8.
Structure and the sign of the cardiovascular model of the external mankind of embodiment 5.
Obtain as described in example 1 above the HUVEC and the hASC that use in the present embodiment.By the myocardial cell in hESC source differentiation 2 weeks as described in (the same) such as Mummery.To beat and bunch cut, separate, and augmenting the DMEM/F12(EB substratum of 10%FBS, 1%NEAA and 1%Glutamax) in cultivate.
As described below in 48 orifice plates construct unfaithful intention vascular pattern:
The 0th day: tubule forms the structure of platform
Coculture model: hASC(is gone down to posterity at most 4 times) with 20000 cell/cm
2density be seeded in the EGM-2BulletKit substratum in 48 orifice plates.After 1-3 hour, maximum the HUVEC(in EGM-2 substratum 4 times are gone down to posterity) with 4000 cell/cm
2density be seeded in carefully on the top of hASC.
Single culture model: hASC(is gone down to posterity at most 4 times) with 24000 cell/cm
2density be seeded in the EGM-2BulletKit substratum in 48 orifice plates.
The 1st day: the structure of external cardiovascular model
Myocardial cell's (hole 1-7 cell aggregation of each 48 orifice plates) in the hESC source in EB is seeded on the top of tubule formation platform.
The 2nd day: the induction of differentiation
Substratum is replaced by the EB that augments 10ng/ml vascular endothelial growth factor (VEGF) and 1ng/ml Prostatropin (FGF-2).Time substratum is replaced with to fresh culture on every Wendesdays.
Result
Fig. 4 shows in coculture model, even mankind myocardial cell also has function after 10 days, shrinkable and show the representative configuration of similar adult cardiomyocytes.
Claims (20)
1. external cardiovascular structures, it comprises:
I) tubule separating forms platform, and described tubule forms platform and comprises mankind's fat stem cell (hASC), and
Ii) myocardial cell.
2. the structure of claim 1, wherein said tubule forms platform and also comprises the endotheliocyte that forms tubule.
3. the structure of claim 2, the endotheliocyte of wherein said formation tubule is selected from endotheliocyte, endotheliocyte, the endothelial progenitor cells in transdifferentiation source and the endotheliocyte obtaining by genetic modification in the multipotential stem cell source of endotheliocyte, the induction in endotheliocyte, the hESC source in human umbilical vein endothelial cell, human microvascular endothelial cell, mankind's fat stem cell source.
4. the structure of claims 1 to 3 any one, wherein said myocardial cell is selected from the myocardial cell in neonatal cardiac myocytes, hiPSC source, the myocardial cell in hESC source, the myocardial cell in adult stem cell source, myocardial cell and the primary myocardial cell of the mankind in transdifferentiation source.
5. the structure of claim 1 to 4 any one, it also comprises the biomaterial of exogenous substrates component or interpolation, the biomaterial of described exogenous substrates component or interpolation is selected from synthetic polymer, natural polymer, collagen protein I, collagen protein IV, hyaluronic acid, gelatin and other extracellular matrix components, or its mixture.
6. the structure of claim 1 to 5 any one, it is used for the treatment of heart trouble.
7. the structure of claim 5, wherein said heart trouble is selected from coronary heart disease and DCM (dilated cardiomyopathy).
8. the tubule separating forms platform, and it comprises mankind's fat stem cell (hASC).
9. the platform of claim 8, it does not contain the biomaterial of any exogenous substrates component or interpolation.
10. the tubule of production claim 8 forms the method for platform, and described method comprises:
A) provide hASC;
B) augmenting in the cell culture medium of VEGF and FGF-2, optionally under the condition of biomaterial that does not have any exogenous substrates component or interpolation, cultivate described hASC.
The method of 11. claims 10, it also comprises: the endotheliocyte that forms tubule is provided, and common cultivation is carried out in they and described hASC.
The method of 12. claims 11, the endotheliocyte of wherein said formation tubule is selected from the endotheliocyte in the multipotential stem cell source of endotheliocyte, the induction in endotheliocyte, the hESC source in human umbilical vein endothelial cell, human microvascular endothelial cell, mankind's fat stem cell source, endotheliocyte and the endothelial progenitor cells in transdifferentiation source.
The method of 13. claim 10 to 12 any one, wherein said substratum also comprises at least one and is selected from following reagent: xitix, hydrocortisone, heparin, IGF-1 and EGF.
The method of the external cardiovascular structures of 14. production claims 1, described method comprises:
A) provide the endotheliocyte of hASC, myocardial cell and optional formation tubule;
B) cultivate described hASC, optionally together with the endotheliocyte of described hASC and described formation tubule, cultivate;
On the top of the culture c) described myocardial cell being formed in step b), cultivate; And
D) use VEGF and FGF-2 to the cell culture forming in step c).
The method of 15. claims 14, the endotheliocyte of wherein said formation tubule is selected from the endotheliocyte in the multipotential stem cell source of endotheliocyte, the induction in endotheliocyte, the hESC source in human umbilical vein endothelial cell, human microvascular endothelial cell, mankind's fat stem cell source, endotheliocyte and the endothelial progenitor cells in transdifferentiation source.
The method of 16. claims 14 or 15, wherein step c) also comprises that using at least one is selected from following reagent: xitix, hydrocortisone, heparin, IGF-1 and EGF.
17. measure the bioactive method of tested substance, and described method comprises the following steps:
A) provide the external cardiovascular structures of claim 1 or the tubule of claim 8 to form platform;
B) use described tested substance to described structure or platform;
C) in described structure or platform, measure the effect of described tested substance; And
D) by the effect of measuring in step c) with do not exist the respective effects of measuring under the condition of described tested substance to compare.
The method of 18. claims 17, wherein biological activity to be determined is selected from cytotoxicity, tubule forms and regulates activity, the speed regularity of for example mycardial contractility of electrical properties, repolarization time length, the existence of arrhythmogenic, mechanical properties for example mycardial contractility power and cellular metabolism.
19. treat cardiopathic method in the patient of needs, and described method comprises the cardiac structure of claim 5 is implanted in described patient.
The method of 20. claims 19, wherein said heart trouble is selected from coronary heart disease and DCM (dilated cardiomyopathy).
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105838670A (en) * | 2015-01-13 | 2016-08-10 | 上海交通大学医学院附属第九人民医院 | Cell mixture and preparation method and application thereof |
| CN105911096B (en) * | 2016-03-29 | 2018-07-10 | 南京艾尔普再生医学科技有限公司 | A kind of artificial heart system that can carry out drug pharmacological toxicology screening in vitro |
| CN111793609A (en) * | 2020-09-08 | 2020-10-20 | 北京达熙生物科技有限公司 | Method for promoting proliferation and differentiation of adipose-derived stem cells |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104940997A (en) * | 2014-03-27 | 2015-09-30 | 复旦大学 | Human tissue-engineered cardiac muscle tissue |
| US20190017029A1 (en) * | 2016-02-25 | 2019-01-17 | The J. David Gladstone Institutes, a testament trust established under the Will of J. David | Generation of Expandable Cardiovascular Progenitor Cells |
| IT201800007946A1 (en) * | 2018-08-07 | 2020-02-07 | 1Lab Sa | Model to simulate the behavior of dysfunctional vessels in-vitro |
| US20220170911A1 (en) * | 2019-04-01 | 2022-06-02 | Toppan Inc. | Cell construct and cell construct production method |
| WO2022113540A1 (en) * | 2020-11-26 | 2022-06-02 | 凸版印刷株式会社 | Method for producing tissue body and method for promoting differentiation of fat-derived stem cells |
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- 2012-06-14 KR KR1020147000113A patent/KR20140048190A/en not_active Application Discontinuation
- 2012-06-14 US US14/128,766 patent/US20140206029A1/en not_active Abandoned
- 2012-06-14 CN CN201280030909.0A patent/CN103814124A/en active Pending
- 2012-06-14 EP EP12803040.0A patent/EP2723853B1/en not_active Not-in-force
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| CN105911096B (en) * | 2016-03-29 | 2018-07-10 | 南京艾尔普再生医学科技有限公司 | A kind of artificial heart system that can carry out drug pharmacological toxicology screening in vitro |
| CN111793609A (en) * | 2020-09-08 | 2020-10-20 | 北京达熙生物科技有限公司 | Method for promoting proliferation and differentiation of adipose-derived stem cells |
Also Published As
| Publication number | Publication date |
|---|---|
| CA2839052A1 (en) | 2012-12-27 |
| BR112013033246A2 (en) | 2017-03-01 |
| EP2723853B1 (en) | 2017-12-27 |
| WO2012175797A1 (en) | 2012-12-27 |
| KR20140048190A (en) | 2014-04-23 |
| JP2014519837A (en) | 2014-08-21 |
| EP2723853A4 (en) | 2015-07-22 |
| US20140206029A1 (en) | 2014-07-24 |
| DK2723853T3 (en) | 2018-04-16 |
| FI20115670A0 (en) | 2011-06-23 |
| EP2723853A1 (en) | 2014-04-30 |
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